Investigation and identification of functional post-translational modification sites associated with drug binding and protein-protein interactions
- Min-Gang Su†1,
- Julia Tzu-Ya Weng†1,
- Justin Bo-Kai Hsu†2,
- Kai-Yao Huang1, 3,
- Yu-Hsiang Chi1 and
- Tzong-Yi Lee1, 4Email author
© The Author(s). 2017
Published: 21 December 2017
Protein post-translational modification (PTM) plays an essential role in various cellular processes that modulates the physical and chemical properties, folding, conformation, stability and activity of proteins, thereby modifying the functions of proteins. The improved throughput of mass spectrometry (MS) or MS/MS technology has not only brought about a surge in proteome-scale studies, but also contributed to a fruitful list of identified PTMs. However, with the increase in the number of identified PTMs, perhaps the more crucial question is what kind of biological mechanisms these PTMs are involved in. This is particularly important in light of the fact that most protein-based pharmaceuticals deliver their therapeutic effects through some form of PTM. Yet, our understanding is still limited with respect to the local effects and frequency of PTM sites near pharmaceutical binding sites and the interfaces of protein-protein interaction (PPI). Understanding PTM’s function is critical to our ability to manipulate the biological mechanisms of protein.
In this study, to understand the regulation of protein functions by PTMs, we mapped 25,835 PTM sites to proteins with available three-dimensional (3D) structural information in the Protein Data Bank (PDB), including 1785 modified PTM sites on the 3D structure. Based on the acquired structural PTM sites, we proposed to use five properties for the structural characterization of PTM substrate sites: the spatial composition of amino acids, residues and side-chain orientations surrounding the PTM substrate sites, as well as the secondary structure, division of acidity and alkaline residues, and solvent-accessible surface area. We further mapped the structural PTM sites to the structures of drug binding and PPI sites, identifying a total of 1917 PTM sites that may affect PPI and 3951 PTM sites associated with drug-target binding. An integrated analytical platform (CruxPTM), with a variety of methods and online molecular docking tools for exploring the structural characteristics of PTMs, is presented. In addition, all tertiary structures of PTM sites on proteins can be visualized using the JSmol program.
Resolving the function of PTM sites is important for understanding the role that proteins play in biological mechanisms. Our work attempted to delineate the structural correlation between PTM sites and PPI or drug-target binding. CurxPTM could help scientists narrow the scope of their PTM research and enhance the efficiency of PTM identification in the face of big proteome data. CruxPTM is now available at http://csb.cse.yzu.edu.tw/CruxPTM/.
Proteins are the major functional molecules in living cells, playing essential roles in various cellular processes such as catalysis, transport, and structural integrity. Although the human genome is estimated to harbor approximately 25,000 genes , alternative splicing of transcripts and post-translational modifications (PTMs) of proteins result in millions of proteins with diverse functions [2, 3]. PTMs regulate a protein’s function, level, and activity through the covalent attachment of small chemical molecules to certain amino acid residues, allowing proteins to respond to developmental signals or environmental stimuli [4, 5]. A protein’s structure can also be altered by these site-specific chemical modifications, leading to changes in stability, localization, and associations with other interacting molecules .
Number of PTM proteins associated with PPI and drug binding
PPI annotation (string)
PTM & PPI proteins
PTM & drug-binding proteins
Indeed, an increasing number of studies are uncovering evidence of PTMs regulating drug-target interactions. For example, the epigenetic regulation of the chaperone cycle in different cell types or environmental conditions is found to involve changes in Hsp90 (heat shock protein 90) function through PTM . Moreover, it has been shown that the effect of Hsp90 inhibitors could be enhanced when enzymes that facilitate the PTM of Hsp90 were suppressed , lending support to PTM being a potential therapeutic strategy for modulating the activity of Hsp90 in cancer cells. Phosphorylation, a common PTM of proteins, has also been utilized in drug-target design, whereby the interaction between the drug and the target is controlled by the state of phosphorylation [23, 24]. For instance, it is suggested that various upstream activators and different phosphorylation states can have a spectrum of effects on MEK inhibition, and therefore, greatly influence drug-target interaction with respect to MEK kinase pathway . Since a large proportion of proteins undergo PTMs, it is likely that changes in PTMs regulate a drug’s efficacy and interaction with its target. Also, (Additional file 1: Table S1) shows that drugs can be categorized into two classes, small molecule drugs and biologics . In general, most drugs are considered small organic compounds with a low molecular weight of less than 900 Da. Thus, for PTM studies in the context of drug-target binding, it would be reasonable to focus on the effects, frequency, and location of PTM near the site of binding by small molecule drugs.
The function of a protein can also be regulated by non-covalent PPIs [5, 27–31], a type of highly specific physical interactions between two or more protein molecules . Many cellular processes are carried out through the complex interactions between various proteins, making up the interactome of a living cell or an organism. The binding affinities of these interacting proteins are also regulated by PTMs . According to PTM data on the dbPTM database, more than 60% of PTM sites are found in the domains of proteins that actively participate in PPIs , providing support for a connection between PTM and PPI, and revealing the functions of the proteins involved in PPI. Thus, it is reasonable to assume that proteins capable of undergoing specific PTMs may exhibit certain properties related to PPI.
The diverse effects of PTMs on proteins, as well as their regulatory functions in various cellular processes contributed to the focus of this study—the investigation of drug-target binding and PPIs associated with PTM sites. In particular, we integrated protein tertiary structure and PPI information with the associated PTM sites from the annotations of 3did (3D interacting domains) . PTM peptides were manually curated, and based on their sequence identity with records in the Protein Data Bank (PDB)  and UnitProtKB ID, mapped to their associated proteins. To uncover the impact on binding attributable to residues structurally surrounding PTM substrate, we investigated the orientations of side chains encompassing these neighboring residues in relation to the location of the PTM substrate sites in a protein structure. Finally, we constructed a database-assisted system, CruxPTM, to provide comprehensive information regarding PTM sites on protein tertiary structures, including the site-specific spatial composition of residues, surface area that is accessible to solvent, and residues that surround the PTM sites.
Mapping of PTM sites to the tertiary structures of proteins
To identify the spatial composition of PTM substrate sites within the tertiary structures of proteins, we obtained from the PDB protein structures that have been determined by NMR or X-ray crystallography with an experimental resolution less than 2.5 Å . According to the annotations in UniProtKB, 23,605 proteins in the PDB have 3D structure information. Also, chemical groups that can be covalently attached to the side chain of target residues were observed only in a few protein structures. Thus, to locate PTM substrate sites in 3D, mapping was performed between experimentally verified PTM peptides and the PDB protein records, and cross-referenced with the annotations of tertiary structures on UniProtKB with 100% similarity in sequence identity. Additionally, PTM sites possessing protein structures with modified residues were obtained from annotations on the PTM-SD database . Most PTM sites that are mapped to structural sites are presented in the unmodified state, but PTM-SD provides complete information for modified PTM sites in 3D structures.
Investigation of PTM sites associated with drugs binding
While it is suggested that the binding affinity of a small molecule can be regulated by a phosphorylation site within 12 Å of the site of binding , there is still a lack of information regarding the occurrences and influence of PTMs near drug-target binding. Therefore, we proposed a method in this study to identify PTM sites involved in drug binding. Figure 1 illustrates the workflow for extracting sites of drug-target binding in protein 3D structure. The entire process can be divided into two steps: 1) the processing of experimentally verified binding sites, and 2) molecular docking of drug binding. In step 1, we collected the structural information of small molecules that have associated keywords such as “drug,” “inhibitor,” “agonist” or “antagonist” and have drug annotations in the DrugBank . A total of 34,555 PDB structures and 4803 small drug molecules which have DrugBank annotations were obtained. Then, the PoseView  method was employed to check the binding sites of each drug in the target proteins. PoseView provides a two-dimensional (2D) diagram showing how the drug ligand and the amino acid residues of the target protein may be arranged at the site of interaction. The nature of the interaction is presented in three ways. Black dashed lines indicate hydrogen bonds, salt bridges, and metal interactions. Green solid lines show hydrophobic interactions and green dashed lines represent π-π and π-cation interactions.
In step 2, a docking program, iGEMDOCK 2.0 , was utilized for the computational extraction of drug binding sites. We followed the four sequential steps in iGEMDOCK to perform the drug-target interaction analysis: target and database preparations, molecular docking and post-docking analyses. First, coordinates of the target protein atoms acquired from PDB, the ligand binding area, the atom’s formal charge and the atom types were specified. This procedure allowed iGEMDOCK to read the atom coordinates of a ligand from the prepared ligand database. After the ligand database and the target proteins were determined, docking was analyzed for each ligand using the flexible docking function provided by iGEMDOCK. The final step constituted the re-ranking and sorting of all docked ligand conformations based on an empirical scoring function and an evolutionary approach. The output of the program consisted of details regarding the docking result of each binding site, as well as the atomic characteristics of the target residues that interact with a specific drug ligand by hydrogen bonding (H), electrostatic (E) and van der Waal contact (V). A total of 1991 approved drugs from the DrugBank with 1632 target proteins were investigated by this proposed method. After mapping the experimentally verified PTM sites to the PDB structures, the PTM sites located in a drug binding site were determined to have strong associations with drug-binding, while those in the side chains that were within 12 Å of a drug-binding site were considered to be have relatively weak association with drug binding.
Identification of PTM sites related to protein-protein interactions
In this work, the information of protein functional domains and PPIs were integrated for the identification of PTM-dependent protein interactions. To investigate the preferred functional domains of PTMs, we extracted the domain annotations from the Pfam database, which gives protein “signatures” based on protein families, domains and functional sites. In order to comprehensively study the structural properties of PTM sites associated with protein-interaction domains, the 3D structures of PPIs were acquired from the PDB. By adopting the 3D Interacting Domains (3DID) method proposed by Mosca et al. , the interaction interface of domain-domain interactions in the PDB 3D structures were determined as illustrated in Fig. 1. First we searched for protein structures with more than two subunits, and calculated the number of contact residues on the interface of the Pfam domain region containing the two subunits. Next, we applied a method based on previously published literature by Aloy and Russell , in which they derived the main-chain to side-chain and side-chain to side-chain potentials from the type of complexes described above. In particular, Aloy and Russell  defined interacting residues by using one or more of the following properties: hydrogen bonds (N-O distance of 3.5 Å), salt bridges (N-O distance of 5.5 Å), or van de Waals interactions (C-C distance of 5 Å). If there exists more than five pairs of residue contacts between two domains of a two-subunit region, these two subunits would be defined as an interaction structure. The contact residues were also extracted. A total of 30,455 PDB structures and 13,645 proteins were analyzed and 15,124 protein-protein interaction pairs were defined. Mapping between the experimentally verified PTM sites and the PDB structure uncovered PTM sites located on the PPI interfaces. These sites were regarded as PTM-driven PPIs.
Results and discussion
PTM substrate site characterization
Case study of PTM sites associated with drug binding
Number of PTM sites associated with drug binding sites
Number of PTM sites located on drug contact sites
Number of PTM sites within 12 Å distance of drug contact sites
Pyrrolidone carboxylic acid
Functions of PTM sites on protein-protein interactions
As shown in (Additional file 3: Table S3), of all the experimentally verified PTM sites, over 20% can be found in the functional domains of proteins, implicating the biological significance of PTMs. We studied these sites to infer the roles that these PTMs play in PPI interactions. For instance, approximately 70% of known S-nitrosylation sites, which are responsible for the regulation of NO-related cellular processes, are located in functional domains. Also, among the data that we have collected for the current study, more than 1900 PTM sites are localized to the interface of domain-domain interacting regions. Based on our observations, it appears that structural associations exist between many PTM sites and binding sites for specific PPI domains and perhaps even regulate the interactions between proteins by modifying the sites of contact.
In this study, we first mapped PTM sites to the 3D structures of proteins, and adopted multiple methods to describe the structural characteristics of PTM sites in tertiary structures. Already, studies are emerging that use similar methods to investigate PTM; for example, Karabulut and Frishman’s study  that utilizes spatial amino acid composition to identify acetylation sites. However, by employing several different approaches and considering several structural characteristics of a variety of PTM sites associated with drug-target binding and PPI, this work can effectively facilitate the functional study of various types of PTM. Indeed, the reliability of our analysis can be supported by the fact that other studies also identified some of the drug-binding and PPI associated PTM sites uncovered in our investigation.
Our approach has the potential to be applied on drug design, which often centers around the influence of amino acid mutation on the effect of a drug. However, PTMs are also affected by changes in the amino acid sequence. Our study indicates that PTMs can be crucial to a drug’s effect on a structural level, and knowing PTM sites associated with protein-protein interaction is helpful for understanding the biological mechanisms involving these PTM sites.
For situations where information regarding the protein’s structure is lacking, we attempted to overcome this limitation with molecular docking. According to the latest statistics from the PDB in 2016, there are over 122,000 records for protein structures. Although the number of annotated PDB structures is increasing rapidly, information of structural proteins is still limited. When cross-referenced with annotations on UniProtKB, it was found that only 23,605 out of 551,705 reviewed proteins and 12,165 out of 114,895 PTM proteins have crystal structure information, respectively. Some proteins only have partially annotated crystal structure related to specific fragments in their sequences such that it was impossible to map the PTM sites to these proteins’ 3D structures. For example, the ankyrin-3 protein have 16 experimental and 17 putative PTM sites within its sequence of 4377 amino acids, but only the region between amino acid 4088 and 4199 has annotated crystal structure. As a result, only one PTM site could be mapped to this structure. This limitation may affect the reliability of comparison among PTM sites.
CruxTPM is a novel, integrative web platform for the analysis of PTMs and their biological roles in a 3D structural context. It enables the structural characterization and 3D visualization of PTM sites, as well as the investigation of their relationship with drug-target binding and PPI. The tool also provides interactive function like drug structure search, PTM modified structure visualization, online small molecule docking, etc. We hope this study and analytical platform can help enhance the understanding of the biological mechanisms associated with PTMs and improve the efficiency of drug design.
Publication charge for this work was funded by the Ministry of Science and Technology (MOST) of Taiwan under contract numbers of MOST 104-2221-E-155-036-MY2 and MOST 106-2221-E-155-063 to TYL.
Availability of data and materials
The datasets used and analysed during the current study available from the corresponding author on reasonable request.
About this supplement
This article has been published as part of BMC Systems Biology Volume 11 Supplement 7, 2017: 16th International Conference on Bioinformatics (InCoB 2017): Systems Biology. The full contents of the supplement are available online at https://bmcsystbiol.biomedcentral.com/articles/supplements/volume-11-supplement-6.
TYL and JTYW conceived and designed the experiments. MGS, JBKH, and YHC performed the experiments. MGS and KYH analyzed the data. MGS and JBKH wrote the manuscript with revision by TYL and JTYW. All authors read and approved the final manuscript.
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